Field evaluation of transgenic hybrid poplars with desirable wood properties and enhanced growth for biofuel production by bicistronic expression of PdGA20ox1 and PtrMYB3 in wood-forming tissue

Background To create an ideotype woody bioenergy crop with desirable growth and biomass properties, we utilized the viral 2A-meidated bicistronic expression strategy to express both PtrMYB3 (MYB46 ortholog of Populus trichocarpa, a master regulator of secondary wall biosynthesis) and PdGA20ox1 (a GA20-oxidase from Pinus densiflora that produces gibberellins) in wood-forming tissue (i.e., developing xylem). Results Transgenic Arabidopsis plants expressing the gene construct DX15::PdGA20ox1-2A-PtrMYB3 showed a significant increase in both stem fresh weight (threefold) and secondary wall thickening (1.27-fold) relative to wild-type (WT) plants. Transgenic poplars harboring the same gene construct grown in a greenhouse for 60 days had a stem fresh weight up to 2.6-fold greater than that of WT plants. In a living modified organism (LMO) field test conducted for 3 months of active growing season, the stem height and diameter growth of the transgenic poplars were 1.7- and 1.6-fold higher than those of WT plants, respectively, with minimal adverse growth defects. Although no significant changes in secondary wall thickening of the stem tissue of the transgenic poplars were observed, cellulose content was increased up to 14.4 wt% compared to WT, resulting in improved saccharification efficiency of the transgenic poplars. Moreover, enhanced woody biomass production by the transgenic poplars was further validated by re-planting in the same LMO field for additional two growing seasons. Conclusions Taken together, these results show considerably enhanced wood formation of our transgenic poplars, with improved wood quality for biofuel production.

Introduction of a leaky stop codon as molecular tool in Chlamydomonas reinhardtii

Expression of proteins in the chloroplast or mitochondria of the model green alga Chlamydomonas reinhardtii can be achieved by directly inserting transgenes into organellar genomes, or through nuclear expression and post-translational import. A number of tools have been developed in the literature for achieving high expression levels from the nuclear genome despite messy genomic integration and widespread silencing of transgenes. Here, recent advances in the field are combined and two systems of bicistronic expression, based on ribosome reinitiation or ribosomal skip induced by a viral 2A sequence, are compared side-by-side. Further, the small subunit of Rubisco (RBCS) was developed as a functional nuclear reporter for successful chloroplast import and restoration of photosynthesis: To be able to combine RBCS with a Venus fluorescent reporter without compromising photosynthetic activity, a leaky stop codon is introduced as a novel molecular tool that allows the simultaneous expression of functional and fluorescently tagged versions of the protein from a single construct.

Enhanced production of recombinant proteins in Corynebacterium glutamicum by construction a bicistronic gene expression system

Background: Corynebacterium glutamicum is a traditional food-grade industrial microorganism， in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. However, the intrinsic disadvantage of low recombinant protein expression level is still difficult to be solved. Here, according to the bacteria-specific polycistronic feature that multiple proteins can be translated in one mRNA, efforts have been made to insert a leading peptide gene upstream of target genes as an expression enhancer, and it is found that this can remarkably improve the expression level of proteins under the control of inducible tac promoter in C. glutamicum. Results: In this research, the Escherichia coli ( E. coli ) tac promoter combined with 24 different fore-cistron sequences were constructed in a bicistronic manner in C. glutamicum. Three strong bicistronic expression vectors were isolated and exhibited high efficiency under different culture conditions. The compatibility of these bicistronic vectors was further validated using six model proteins- aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), RamA (regulator of acetate metabolism), Bovine interferon-a (BoIFN-a), glycoprotein D protein (gD) of infectious bovine rhinotracheitis virus (IBRV) and procollagen type I N-terminal peptide (PINP). All examined proteins were highly expressed compared with the original vector with tac promoter. Large-scale production of PINP was also performed in fed-batch cultivation, and the highest PINP production level was 1.2 g/L. Conclusion: In this study, the strength of the inducible tac promoter for C. glutamicum was improved by screening and inserting fore-cistron sequences in front of the target genes. Those vectors with bicistronic expression patterns have strong compatibility for expressing various heterogeneous proteins in high yield. This new strategy could be used to further improve the performance of inducible promoters, achieving double competence of inducible control and high yield.

Mechanism of IRSp53 inhibition by 14-3-3

Filopodia are precursors of dendritic spines and polarized cell migration. The I-BAR-domain protein IRSp53 is an essential regulator of filopodia dynamics that couples Rho-GTPase signaling to cytoskeleton and membrane remodeling, playing essential roles in neuronal development and cell motility. Here, we describe a mechanism whereby phosphorylation-dependent inhibition of IRSp53 by 14-3-3 counters membrane binding and activation by Cdc42 or downstream cytoskeletal effectors. Phosphoproteomics, quantitative binding studies and crystal structures show that 14-3-3 binds to two pairs of phosphorylation sites in IRSp53. Using bicistronic expression we obtained a heterodimer of IRSp53 in which only one subunit is phosphorylated, and show that each subunit of the IRSp53 dimer independently binds a 14-3-3 dimer. A FRET-sensor assay developed using natively phosphorylated and 14-3-3-binding competent IRSp53 purified from mammalian cells reveals opposite conformational changes in IRSp53 upon binding of activatory (Cdc42, Eps8) vs. inhibitory (14-3-3) inputs.

Transient Expression of a Recombinant Monoclonal Antibody in HEK293T Cells

Background: Monoclonal antibodies (mAbs) are considered the most important and financially successful category of the biopharmaceuticals. Extensive optimization of the expression vector, host system and culture parameters are required for the successful production of active monoclonal antibodies in mammalian cells. In this regards, transient expression enables rapid and cost-effective production of recombinant proteins for initial characterization. Methods: In the present study, an internal ribosome entry site (IRES) based bicistronic expression system has been evaluated for the transient expression of an anti-CD52 monoclonal antibody in mammalian cells. The IRES based bicistronic vector was generated through sequential cloning of the Light chain (LC), IRES, and Heavy chain (HC) in an intermediate vector and transfer of the resulting fragment to the expression vector. Transfection of the HEK293T cells was performed and antibody expression was analyzed in cell culture supernatant. Results: Restriction enzyme analysis indicated successful cloning of the antibody coding unit in the expression vector. Analysis of EGFP expression indicated successful transfection of the HEK293T cells. Production levels of 220 µg/L of antibody were achieved in HEK293T cells during three days of culture. Conclusion: Our results show the convenience and efficiency of the bicistronic expression system for transient expression of the whole monoclonal antibodies in mammalian cells.